3O1 rRNA processing and ribosomes — Types of RNA processingVery few RNA molecules are transcribed directly into the final mature RNA. Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product. RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

4primary transcript mature RNA. Cytoplasm Nucleus or NucleolusRNA processingRomoval of nucleotidesaddition of nucleotides to the 5’- or 3’- endsmodification of certain nucleotidesmature RNA.

5(1) Removal of nucleotides by both endonucleases and exonucleases(2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products.(3) Modification of certain nucleotides on either the base or the sugar moiety.

6O1 rRNA processing and ribosomes — rRNA processing in prokaryoteThere are 7 different operons for rRNA that are dispersed throughout the genome.Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived.Pre-16S rRNAPre-tRNAPre-23S rRNApre-5S rRNAPromotersTerminatorsrRNA operon

7Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequencesRNA folding

8Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosomeRNP complexformation

11O1 rRNA processing and ribosomes — rRNA processing in eukaryotesrRNA in eukaryotes is also generated from a single, long precursor molecule by specific modification and cleavage stepsThe processes are not so well understood

12The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol IPrecursor sizes are different among organisms (yeast: 7000 nt; mammalian nt), and pre-mRNA processing is also slightly different among organism.

133. The precursor containsone copy of the 18S coding region andone copy each of the 5.8S and 28S coding regions, which together are the equivalent of the 23S rRNA in prokaryote4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.

16The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced.Mature rRNAs complex with protein to form RNPs (nucleolus)Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)

17Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced out to generate mature rRNAs.Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme

19O1 rRNA processing and ribosomes — RNPs and their studyCells contain a variety of RNA-protein complexes( RNPs).These can be studied using techniques that help to clarify their structure and function.These include dissociation, re-assembly, electron microscopy, use of antibodies, RNase protection, RNA binding, cross-linking and neutron and X-ray diffraction.The structure and function of some RNPs are quite well characterized.

24Ribosome Structure (2) mRNA is associated with the 30S subunitTwo tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits.The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.

25O1 rRNA processing and ribosomes — Eukaryotic ribosomeslarger and more complex than prokaryotic ribosomes, but with similar structural and functional properties

31O2 tRNA processing, RNase P and ribozymes — RNase PRibonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursorsRNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa.RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

32O2 tRNA processing, RNase P and ribozymes — RibozymesRibozymes are catalytic RNA molecules that can catalyze particular biochemical reactions.RNase P RNA is a ribozyme.RNase P RNA from bacteria is more catalytically active in vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures.Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequencesGroup I introns, such as Tetrahymena intronGroup II introns.

34O3 mRNA processing, hnRNPs and snRNPs — Processing of mRNAProcessing of mRNA: prokaryotesThere is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.Prokaryotic mRNA is degraded rapidly from the 5’ endProcessing of mRNA in eukaryotesIn eukaryotes, mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA).Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs

36O3 mRNA processing, hnRNPs and snRNPs — hnRNPThe hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP)The hnRNP proteins are though to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions

37O3 mRNA processing, hnRNPs and snRNPs — snRNP particlessnRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs.The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6.A large number of snRNP define methylation sites in pre-rRNA.snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap.Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.

38O3 mRNA processing, hnRNPs and snRNPs — 5’ CappingVery soon after RNA Pol II starts making a transcript, and before the RNA chain is more then nt long, the 5’-end is chemically modified.7-methylguanosine is covalently to the 5´ end of pre-mRNA.Linked 5´  5´Occurs shortly after initiation

42RNA polymerase II does not usually terminate at distinct site O3 mRNA processing, hnRNPs and snRNPs — ’ Cleavage and polyadenylationIn most pre-mRNAs, the mature 3’-end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.RNA polymerase II does not usually terminate at distinct sitePre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)~250 AMPs are then added to the 3´ endAlmost all mRNAs have poly(A) tail

45O3 mRNA processing, hnRNPs and snRNPs — Splicingthe process of cutting the pre-mRNA to remove the introns and joining together of the exons is called splicing.it takes place in the nucleus before the mature mRNA can be exported to the cytoplasm.Introns: non-coding sequencesExons: coding sequencesRNA splicing: removal of introns and joining of exonsSplicing mechanism must be precise to maintain open reading frameCatalyzed by spliceosome (RNA + protein)

47Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch siteStep 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.

49Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.

50Spliceosome Catalyzes pre-mRNA splicing in nucleusComposed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the pre-mRNA being assembledU1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.U2 and U6 snRNA are able to catalyze the splicing reaction.

54O3 mRNA processing, hnRNPs and snRNPs — Pre-mRNA methylationThe final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases.The methylations seem to be largely conserved in the mature mRNA.

55O4 Alternative mRNA processing — Alternative processingAlternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.

56O4 Alternative mRNA processing — Alternative poly(A) siteSome pre-mRNAs contain more than one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.

57O4 Alternative mRNA processing — Alternative splicingThe generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways:By using different promotersBy using different poly(A) sitesBy retaining certain intronsBy retaining or removing certain exons

66O4 Alternative mRNA processing — RNA editingAn unusual form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing.Changing RNA sequence (after transcription)

67RNA editing is known to occur in two different situations, with different causes.In mammalian cells there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded. (Base modification:A or C deamination)In trypanosome mitochondria, more widespread changes occur in transcripts of several genes, when bases are systematically added or deleted. (Base U insertion and deletion)

703. Most eukaryotic pre-mRNAs are matured by which of the following modifications to their ends?A capping at the 3’-end cleavage and polyadenylation at the 5'-end.B addition of a GMP to the 5'-end，cleavage and polyadenylation to create the 3'-end.C addition of a guanine residue to the 5'-end cleavage and polyadenylation to create the 3'-end.D addition of a GMP to the 5'-end，polyadenylation，then cleavage to create the 3'-end.4. Which one of the following statements correctly describes the splicing process undergone by most eukaryotic pre-mRNAs?A in a two-step reaction, the spliceosome removes the exon as a lariat and joins the two introns together.B splicing requires conserved sequences which are the 5ιsplice site，the 3' -splice site the branch-point and the polypurine tract.C the U1 snRNP initially binds to the 5'-splice site，U2 to the branchpoint sequence and then the tri-snRNP, U4, US and U6 can bind.D in the first step of splicing the G at the 3'-end of the intron is joined to the 2’-hydroxyl group of the A residue of the branchpoint sequence to create a lariat.